抗汉城病毒荧光单克隆抗体的制备及初步应用

目的 制备抗汉城病毒（Seoul virus，SEOV）荧光单克隆抗体（单抗），并初步应用于Ⅱ型肾综合征出血热灭活疫苗原液的病毒灭活验证。 方法  以SEOV L-99株灭活病毒原液为免疫原，采用小鼠杂交瘤细胞融合技术，筛选抗SEOV特异性单抗杂交瘤；小鼠体内诱生法制备单抗腹水，蛋白A亲和层析纯化单抗，并以蛋白质印迹法鉴定其特异性；间接ELISA测定单抗效价和相对亲和力；纯化单抗采用搅拌法标记异硫氰酸荧光素，并分别采用直接免疫荧光法与小鼠脑内接种法对Ⅱ型肾综合征出血热灭活疫苗原液进行病毒灭活验证。结果 共筛选出4株特异性抗SEOV L-99株杂交瘤1D5、2E3、3A4及5B7。蛋白质印迹法鉴定4株单抗均与SEOV L-99株核衣壳蛋白发生特异性反应；间接ELISA测定腹水效价为3.13×10^-8~ 1.25×10^-7；相对亲和力顺序为1D5>3A4>2E3>5B7；纯化抗体纯度>95%；异硫氰酸荧光素标记1D5株单抗的结合比率为3.4；直接免疫荧光法与小鼠脑内接种法检测盲传3代的Ⅱ型肾综合征出血热灭活疫苗原液，病毒灭活验证结果一致。结论 成功筛选到高效价特异性抗SEOV单抗，并制备成荧光单抗，可用于Ⅱ型肾综合征出血热灭活疫苗生产工艺中的病毒灭活验证。

Preparation and preliminary application of fluorescent monoclonal antibodyagainst Seoul virus

Objective  The prepare fluorescent monoclonal antibody (McAb) against Seoul virus (SEOV) and preliminarily apply to the inactivation verification in the stock solution of type Ⅱ hemorrhagic fever with renal syndrome inactivated vaccine. Methods Using the inactivated virus stock solution of SEOV L-99 strain as immunogen, mouse hybridoma cell fusion technology was used to screen specific anti-SEOV McAb hybridoma. McAb ascites was prepared by induction method in mice and purified by protein A affinity chromatography, and its specificity was identified by Western blot. The titer and relative affinity of McAb were determined by indirect ELISA. The purified McAb was labeled with fluorescein isothiocyanate (FITC) by stirring method, and the virus inactivation validation in the stock solution of type Ⅱ hemorrhagic fever with renal syndrome inactivated vaccine was detected by direct immunofluorescence method and mouse intracerebral inoculation method. Results  Four hybridomas 1D5, 2E3, 3A4 and 5B7 with specific anti-SEOV L-99 activity were screened. Western blot showed that the four McAbs reacted specifically with nucleocapsid protein of SEOV L-99 strain. The titer of ascites determined by indirect ELISA was 3.13×10⁸﹣1.25×10^-7. The relative affinity order of 4 McAbs was 1D5 > 3A4 > 2E3 > 5B7. Purities of purified antibodies were >95%. The binding ratio of FITC label 1D5 McAb was 3.4. The direct immunofluorescence method and mouse intracerebral inoculation method were used to detect inactivated vaccines of type Ⅱ hemorrhagic fever with renal syndrome for three generations blindly, and the results of virus inactivation validation were consistent. Conclusion A high-titer specific anti-SEOV McAb is Successfully screened , and fluorescently labeled, which can be applied to the verification of virus inactivation during the production process of the type Ⅱ hemorrhagic fever with renal syndrome inactivated vaccine.